- Email: [email protected]
Tissue distribution of complement regulatory proteins in rat gastrointestinal tracts
A842
AGA ABSTRACTS
PREVALENCE OF CAG-A GENE AND V A C U O L A T I N G CYTOTOXIN ACTIVITY OF H E L I C O B A C T E R PYLORI ISOLATES FROM PATIENTS WITH ATROPHIC GASTRITIS OR DUODENAL ULCER. Y. Ito, T. Azuma, S.'Ito, M. Hirai, H. Murakita, H. Miyaji, T. Kato, Y. Kohli, and M. Kuriyama. Second Dept. o f Internal Medicine, Fukui Medical School, Fukui, Japan. '~
Helicobacterpylori infection is widely accepted as the predominant of chronic gastritis, and is strongly associated with duodenal ulcer disease. The pathogenic mechanisms whereby H. pylori causes human disease remain poorly understood. Why do only a minority of patients harbbring H. pylori develop duodenal ulcer disease? There may be ulcerogenic strains in the bacterium. At present, the only phenotypic characteristics known to differ among strains are the production of a vacuolating cytotoxin and the presence of a 128-kDa cytotoxin-associated protein encoded by cagA. CagA protein is 'highly immunogenic, and its presence is strongly associated with the cytotoxin production. Since high titers of serum antibodies to the cagA protein has been detected in all patients with duodenal ulcer and most of those with gastric carcinoma, it has been proposed that the disease development requires infection with cytotoxinproducing strains. In the present study, we investigated cytotoxin activities of 1-1.pylori isolates from patients with atrophic gastritis or duodenal ulcer, the prevalence of cagA and vacA genes in the strains, and the correlation among them. Methods: DNA samples ofH. pylori isolates from 35 patients with atrophic gastritis and 25 patients with duodenal ulcer were examined. To detect cagA and vacA genes, polymerase-chain reaction (PCR) was carried out. Based on the published sequence, oligonueleotides of length 20 were prepared to amplify the region Of the sequence: nucleotides 1764-2083 for cagA, nucleotides 768-1164 for vacA. Cytotoxin activities in the culture media which were 20-fold concentrated were semi-quantitatively analyzed using A431 ceils ~is indicator cells. The relative activ,ity ~f vacuolating cytotoxin in a sample was defined as the maximum dilution times. R e s u l t s : The production 0f cytotoxin was observed in 25 (71.4%) out of 35 strains from patients with atrophic gastritis and 15 (60.0%) t u t t i 25 strains from patients wida duodenal ulcer. VagA gene was detected in all samples examined, however, cagA gene was detected in 32 (91.4%) out of 35 Strains from patients with atrophic gastritis and 22 (88.0%) out of 25 strains from patients with duodenal ulcer. Four out of 6 cagA-negative strains presented the vacuolating cytot0xin activity. Conclusions: The present study suggest that cytotoxic:strains are associated more frequently with atrophic gastritis than with duodenal ulcer, and that cagA gene does not affect the cytotoxin activity of H. pylori. cause
TISSUE DISTRIBUTIONOF COMPLEMENTREGULATORYPROTEINS IN RAT GASTROINTESTINALTRACTS. F. Iwata~), M. Itoh, T. Job, T~ Takeuchi2), T. Yamamotol), T. Tadas), N. Okada, H. Okada4), B.P. M0rganS).1) Dept. Med., Nagoya City Kouseiin, 2) 1st Dept. Int. Mad., a) Dept. Pathology, 4) Dept. MoI. Biol., Nagoya City Univ. Sch. Med., Nagoya, Japan, S)Univ. Wales Coll. Med., Cardiff, U.K. Complement regulatory proteins, which regulate the activation of complement ether by acting on C3/C5 convertase of the classical and alternative pathways or by preventing the formation of membrane attack complexes (MAC), may protect complement-mediated injury. However, the importance of these factors has not been well investigated in gastrointestinal tracts. In this study, tissue distribution of these proteins were studied Using monoelonal antibodies 512 which was directed against a rat erythrocyte membrane inhibitor of the C3 converting step (BBRC 1994; 198: 819) and 6D1 against a rat CD59 which inhibited the formation of MAC (Bioohem. J. 1992; 284: 169). Methods: Frozen tissue sections of rat gastrointestinal tracts (esophagus, forestemach, glandular stomach, duodenum, jejunum, ileum, cecum and colon) were cut to 6 gm thickness by cryostat. The sections were treated with the antibodies (512, 6D1). Incubation was carried out with avidin-biotin-peroxidase complex (Histofine kit, Nichirei, Tokyo, Japan). Treated sections were developed with 3,3'-diaminobenzidine in Tris-HCI, pH 7.6, containing H202. After washing in tap water, the sections were eounterstained with methyl green prior to mounting, and were observed under a conventional light microscope. Results: Smooth muscle and endothelial cells in all of gastrointestinal tracts were stained with both of 512 and 6D1. The surface of glandularepithelial cells of glandular stomach, duodenum, jejunum, ileum, ceCum and colon was also stained. In glandular stomach, the epithelial cells of fundic glands were strongly stained. The positive expression of these proteins was also shown in the squamous cells of esophagus and forestomach. (~onclualon and disouaslom The tissue of gastrointestinal tracts was Stained well with antibodies 512 and 6D1. Since 5!2 antigen and CD59 are expressed significantly on the cells of gastrointestinal tracts, prevention of complement at those sites by them will be essential to keep a homeostasis. Therefore, impairment of the function of complement regulatory proteins may induce path.ogenic change and this will be confirmed by local inoculation of 512 and/or 6D1.
GASTROENTEROLOGY, Vol. 1 0 8 , No. 4
A P O P T O S I S O F C R Y P T E P I T H E L I U M IN U L C E R A T I V E C O L I T I S . M. i w a m o t o ~, T . K o j i 2, K. M a k i y a m a ' , P.K. N a k a n e ' . D e p t s . o f M e d i c i n II' & A n a t o m y III ~ , N a g a s a k ! U n i v . Sch. o f M e d . , N a g a s a k i , Japan. U l c e r a t i v e colitis ( U C ) is c h a r a c t e r i z e d by loss o f e p i t h e l i u m a n d e x t e n s i v e i n f l a m m a t i o n . Q u e s t i o n s , ( 1 , ) is t h e loss r e s u l t e d f r o m d e a t h o f e p i t h e l i a l cells? a n d if t h e a n s w e r is "yes", t h e n (2) is it by way o f a p o p t o s i s or n e c r o s i s ? a n d (3) w h e t h e r t h e d e a t h p r e c e d e s or follows t h e o n s e t o f inflammati, o n w e r e asked. M e t h o d s : C o l o n i c b i o p s i e s f r o m n o r m a l a r e a s o f i?atient s wi'th c o l o n polyps, f r o m inflamed areas and from their adjacent uninvolved areas of u n t r e a t e d U C c o l o n were p r o c e s s e d for histological e x a m i n a t i o n . S o m e biopsies were p r 0 c e s s e d for r o u t i n e e l e c t r o n m i c r o s c o p y . F o r i d e n t i f i c a t i o n o f a p o p t o t i c ceils at t h e l i g h t m i c r o s c o p i c level, D N A s t r a n d bre'aks were visualized by in situ nick t r a n s l a t i o n a n d t e r m i n a l d e o x y n u c l e o t i d y l t r a n s f e r a s e r e a c t i o n a n d F a s a n t i g e n was l o c a l i z e d by i m m u n o h i s t o c h e m i s t i y . ResuIts: A p o p t o s i s m a r k e r p o s i t i v e cells were f o u n d m a i n l y o n l u m i n a l , . e p i t h e l i u m o f n o r m a l c o l o n . W h e r e a s , in t h e i n f l a m e d as well as u n i n v o l v e d a r e a s 0 f U C c o l o n i c biopsies, a p o p t o s i s m a r k e r p o s i t i v e cells w e r e f o u n d in crypt as well as l u m i n a l e p i t h e l i a . U l t r a s t r u c t u a l f e a t u r e s o f a p o p t o s i s were f o u n d in t h e s e p o s i t i v e Ceils. C o n c l u s i o n s : T h e s e f i n d i n g s s u g g e s t t h a t s o m e e p i t h e l i a l cells in crypt die o.f a p o p t o s i s p r i o r to t h e o n s e t o f i n f l a m m a t t h i n U C c o l o n a n d t h e ' ' i n f l a m m a t i o n is S e c o n d a r y to ,the loss o f crypt e p i t h e l i u m l
MODULATORY EFFECT OF T LYMPHOCYTES ON EXPERIMENTAL COLITIS IN RATS. K. Jacobson and S.M. Collins. Intestinal Diseases Research Programme, McMaster University, Hamilton, Ontario, Canada. A defect in immune regulation is postulated to be an important factor in the expression of inflammatory bowel disease in man. In rats, acute colitis develops following intrarectal administration of trinitrobenzene sulfonic acid (TNB) plus ethaiiol. Since TNB is a hapten, the involvement of an immunological component has been postulated in the pathogeuesis of this model of colitis. In this study, we examined the role o f t lymphocytes m the expression of colitis following intrarectal administration of TNB. The activity of myeloperoxidase (MPO) in units/mg of tissue was used to monitor the acute inflammatory response in the distal and transverse colon. Studies were performed on congenitally athymic (rnu/ruu) rats and their thymus-bearing littermates (rnu/*). Distal colitis was induced by ir administration of TNB (30 mg in 0.25 mL 50% ethanol) and studies were conducted on day 5 post TNB. In Controls that did not receive TNB, athymic rats exhibited higher levels of MPO activity in the distal colbn (3.6+ 1.29 vs 0.22+0.2 U/rag, P < 0.05) than in thymus-bearing littermates. Administration of TNB to euthymic rats caused a significant increase in MPO activity from 0.22+0.2 to 31.7+16.5 and there were no deaths in this group. In contrast, in athymic rats TNB increased MPO activity from 3.6+1.29 to 182+16.6 and there was an attendant 25% mortality in this group. When animals were examined 6 weeks after induction of colitis, MPO values were similar to those found in the respective control groups in both athymics (15.76+5.6 for TNB vs 12.87_ 1.14 for controls; P > 0.05) and euthymics (0.46+0.14 for TNB vs 0.476+0.01 for controls; P>0.05). These resuks indicate that MPO activity is higher in the distal colon of athymic compared to euthymic rats in the absence of TNB treatment. suggesting a larger presence of resident inflammatory cells in that tissue, and this increased over 6 weeks. Following TNB, the severity, but not the extent or duration, of acute colitis was significantly worse in athymic rats, and had a significant mortality. These results suggest that T lymphocytes play a modulatory anti-inflammatory role in TNB-induced colitis in rats. Supported by MRC Canada.
AGA ABSTRACTS
PREVALENCE OF CAG-A GENE AND V A C U O L A T I N G CYTOTOXIN ACTIVITY OF H E L I C O B A C T E R PYLORI ISOLATES FROM PATIENTS WITH ATROPHIC GASTRITIS OR DUODENAL ULCER. Y. Ito, T. Azuma, S.'Ito, M. Hirai, H. Murakita, H. Miyaji, T. Kato, Y. Kohli, and M. Kuriyama. Second Dept. o f Internal Medicine, Fukui Medical School, Fukui, Japan. '~
Helicobacterpylori infection is widely accepted as the predominant of chronic gastritis, and is strongly associated with duodenal ulcer disease. The pathogenic mechanisms whereby H. pylori causes human disease remain poorly understood. Why do only a minority of patients harbbring H. pylori develop duodenal ulcer disease? There may be ulcerogenic strains in the bacterium. At present, the only phenotypic characteristics known to differ among strains are the production of a vacuolating cytotoxin and the presence of a 128-kDa cytotoxin-associated protein encoded by cagA. CagA protein is 'highly immunogenic, and its presence is strongly associated with the cytotoxin production. Since high titers of serum antibodies to the cagA protein has been detected in all patients with duodenal ulcer and most of those with gastric carcinoma, it has been proposed that the disease development requires infection with cytotoxinproducing strains. In the present study, we investigated cytotoxin activities of 1-1.pylori isolates from patients with atrophic gastritis or duodenal ulcer, the prevalence of cagA and vacA genes in the strains, and the correlation among them. Methods: DNA samples ofH. pylori isolates from 35 patients with atrophic gastritis and 25 patients with duodenal ulcer were examined. To detect cagA and vacA genes, polymerase-chain reaction (PCR) was carried out. Based on the published sequence, oligonueleotides of length 20 were prepared to amplify the region Of the sequence: nucleotides 1764-2083 for cagA, nucleotides 768-1164 for vacA. Cytotoxin activities in the culture media which were 20-fold concentrated were semi-quantitatively analyzed using A431 ceils ~is indicator cells. The relative activ,ity ~f vacuolating cytotoxin in a sample was defined as the maximum dilution times. R e s u l t s : The production 0f cytotoxin was observed in 25 (71.4%) out of 35 strains from patients with atrophic gastritis and 15 (60.0%) t u t t i 25 strains from patients wida duodenal ulcer. VagA gene was detected in all samples examined, however, cagA gene was detected in 32 (91.4%) out of 35 Strains from patients with atrophic gastritis and 22 (88.0%) out of 25 strains from patients with duodenal ulcer. Four out of 6 cagA-negative strains presented the vacuolating cytot0xin activity. Conclusions: The present study suggest that cytotoxic:strains are associated more frequently with atrophic gastritis than with duodenal ulcer, and that cagA gene does not affect the cytotoxin activity of H. pylori. cause
TISSUE DISTRIBUTIONOF COMPLEMENTREGULATORYPROTEINS IN RAT GASTROINTESTINALTRACTS. F. Iwata~), M. Itoh, T. Job, T~ Takeuchi2), T. Yamamotol), T. Tadas), N. Okada, H. Okada4), B.P. M0rganS).1) Dept. Med., Nagoya City Kouseiin, 2) 1st Dept. Int. Mad., a) Dept. Pathology, 4) Dept. MoI. Biol., Nagoya City Univ. Sch. Med., Nagoya, Japan, S)Univ. Wales Coll. Med., Cardiff, U.K. Complement regulatory proteins, which regulate the activation of complement ether by acting on C3/C5 convertase of the classical and alternative pathways or by preventing the formation of membrane attack complexes (MAC), may protect complement-mediated injury. However, the importance of these factors has not been well investigated in gastrointestinal tracts. In this study, tissue distribution of these proteins were studied Using monoelonal antibodies 512 which was directed against a rat erythrocyte membrane inhibitor of the C3 converting step (BBRC 1994; 198: 819) and 6D1 against a rat CD59 which inhibited the formation of MAC (Bioohem. J. 1992; 284: 169). Methods: Frozen tissue sections of rat gastrointestinal tracts (esophagus, forestemach, glandular stomach, duodenum, jejunum, ileum, cecum and colon) were cut to 6 gm thickness by cryostat. The sections were treated with the antibodies (512, 6D1). Incubation was carried out with avidin-biotin-peroxidase complex (Histofine kit, Nichirei, Tokyo, Japan). Treated sections were developed with 3,3'-diaminobenzidine in Tris-HCI, pH 7.6, containing H202. After washing in tap water, the sections were eounterstained with methyl green prior to mounting, and were observed under a conventional light microscope. Results: Smooth muscle and endothelial cells in all of gastrointestinal tracts were stained with both of 512 and 6D1. The surface of glandularepithelial cells of glandular stomach, duodenum, jejunum, ileum, ceCum and colon was also stained. In glandular stomach, the epithelial cells of fundic glands were strongly stained. The positive expression of these proteins was also shown in the squamous cells of esophagus and forestomach. (~onclualon and disouaslom The tissue of gastrointestinal tracts was Stained well with antibodies 512 and 6D1. Since 5!2 antigen and CD59 are expressed significantly on the cells of gastrointestinal tracts, prevention of complement at those sites by them will be essential to keep a homeostasis. Therefore, impairment of the function of complement regulatory proteins may induce path.ogenic change and this will be confirmed by local inoculation of 512 and/or 6D1.
GASTROENTEROLOGY, Vol. 1 0 8 , No. 4
A P O P T O S I S O F C R Y P T E P I T H E L I U M IN U L C E R A T I V E C O L I T I S . M. i w a m o t o ~, T . K o j i 2, K. M a k i y a m a ' , P.K. N a k a n e ' . D e p t s . o f M e d i c i n II' & A n a t o m y III ~ , N a g a s a k ! U n i v . Sch. o f M e d . , N a g a s a k i , Japan. U l c e r a t i v e colitis ( U C ) is c h a r a c t e r i z e d by loss o f e p i t h e l i u m a n d e x t e n s i v e i n f l a m m a t i o n . Q u e s t i o n s , ( 1 , ) is t h e loss r e s u l t e d f r o m d e a t h o f e p i t h e l i a l cells? a n d if t h e a n s w e r is "yes", t h e n (2) is it by way o f a p o p t o s i s or n e c r o s i s ? a n d (3) w h e t h e r t h e d e a t h p r e c e d e s or follows t h e o n s e t o f inflammati, o n w e r e asked. M e t h o d s : C o l o n i c b i o p s i e s f r o m n o r m a l a r e a s o f i?atient s wi'th c o l o n polyps, f r o m inflamed areas and from their adjacent uninvolved areas of u n t r e a t e d U C c o l o n were p r o c e s s e d for histological e x a m i n a t i o n . S o m e biopsies were p r 0 c e s s e d for r o u t i n e e l e c t r o n m i c r o s c o p y . F o r i d e n t i f i c a t i o n o f a p o p t o t i c ceils at t h e l i g h t m i c r o s c o p i c level, D N A s t r a n d bre'aks were visualized by in situ nick t r a n s l a t i o n a n d t e r m i n a l d e o x y n u c l e o t i d y l t r a n s f e r a s e r e a c t i o n a n d F a s a n t i g e n was l o c a l i z e d by i m m u n o h i s t o c h e m i s t i y . ResuIts: A p o p t o s i s m a r k e r p o s i t i v e cells were f o u n d m a i n l y o n l u m i n a l , . e p i t h e l i u m o f n o r m a l c o l o n . W h e r e a s , in t h e i n f l a m e d as well as u n i n v o l v e d a r e a s 0 f U C c o l o n i c biopsies, a p o p t o s i s m a r k e r p o s i t i v e cells w e r e f o u n d in crypt as well as l u m i n a l e p i t h e l i a . U l t r a s t r u c t u a l f e a t u r e s o f a p o p t o s i s were f o u n d in t h e s e p o s i t i v e Ceils. C o n c l u s i o n s : T h e s e f i n d i n g s s u g g e s t t h a t s o m e e p i t h e l i a l cells in crypt die o.f a p o p t o s i s p r i o r to t h e o n s e t o f i n f l a m m a t t h i n U C c o l o n a n d t h e ' ' i n f l a m m a t i o n is S e c o n d a r y to ,the loss o f crypt e p i t h e l i u m l
MODULATORY EFFECT OF T LYMPHOCYTES ON EXPERIMENTAL COLITIS IN RATS. K. Jacobson and S.M. Collins. Intestinal Diseases Research Programme, McMaster University, Hamilton, Ontario, Canada. A defect in immune regulation is postulated to be an important factor in the expression of inflammatory bowel disease in man. In rats, acute colitis develops following intrarectal administration of trinitrobenzene sulfonic acid (TNB) plus ethaiiol. Since TNB is a hapten, the involvement of an immunological component has been postulated in the pathogeuesis of this model of colitis. In this study, we examined the role o f t lymphocytes m the expression of colitis following intrarectal administration of TNB. The activity of myeloperoxidase (MPO) in units/mg of tissue was used to monitor the acute inflammatory response in the distal and transverse colon. Studies were performed on congenitally athymic (rnu/ruu) rats and their thymus-bearing littermates (rnu/*). Distal colitis was induced by ir administration of TNB (30 mg in 0.25 mL 50% ethanol) and studies were conducted on day 5 post TNB. In Controls that did not receive TNB, athymic rats exhibited higher levels of MPO activity in the distal colbn (3.6+ 1.29 vs 0.22+0.2 U/rag, P < 0.05) than in thymus-bearing littermates. Administration of TNB to euthymic rats caused a significant increase in MPO activity from 0.22+0.2 to 31.7+16.5 and there were no deaths in this group. In contrast, in athymic rats TNB increased MPO activity from 3.6+1.29 to 182+16.6 and there was an attendant 25% mortality in this group. When animals were examined 6 weeks after induction of colitis, MPO values were similar to those found in the respective control groups in both athymics (15.76+5.6 for TNB vs 12.87_ 1.14 for controls; P > 0.05) and euthymics (0.46+0.14 for TNB vs 0.476+0.01 for controls; P>0.05). These resuks indicate that MPO activity is higher in the distal colon of athymic compared to euthymic rats in the absence of TNB treatment. suggesting a larger presence of resident inflammatory cells in that tissue, and this increased over 6 weeks. Following TNB, the severity, but not the extent or duration, of acute colitis was significantly worse in athymic rats, and had a significant mortality. These results suggest that T lymphocytes play a modulatory anti-inflammatory role in TNB-induced colitis in rats. Supported by MRC Canada.